The present invention relates to gene entrapment vectors and their use in gene discovery, and their use in screening for or making cells and organisms that are mutated for such genes. This invention also relates to the use of such entrapment vectors to identify tissue specific transcription control elements such as promoters and enhancers and for generating transgenic animals displaying restricted expression of transgenes. This invention also relates to trap vectors comprising a splice acceptor and a sequence encoding a reporter gene.
Genomic based drug discovery is largely dependent upon the identification of specific genomic targets. Thus, cloning, sequencing, and identification of function of mammalian genes is a first priority in a genomic based drug discovery. In particular, it is important to identify and make use of genes which are spacially and/or temporally regulated in the organism.
Animal model systems such as the fruit fly and the worm are often used in gene identification because of ease of manipulation of the genome and ability screen for mutants. While these systems have their limitations, large numbers of developmental mutations have been identified in those organisms either by monitoring the phenotypic effects of mutations or by screening for expression of reporter genes incorporated into developmentally regulated genes.
Many features of the mouse make it the best animal model system to study gene function. However, the mouse has not been used for large scale classical genetic mutational analysis because random mutational screening and analysis is very cumbersome and expensive due to long generation times and maintenance costs.
A disadvantage in using animal models for the identification of genes is the need to establish a transgenic animal line for each mutational event. This disadvantage is alleviated in part by using embryonic stem (ES) cell lines because mutational events may be screened in vitro prior to generating an animal. ES cells are totipotent cells isolated from the inner cell mass of the blastocyst. Methods are well known for obtaining ES cells, incorporating genetic material into ES cells, and promotion of differentiation of ES cells. ES cells may be caused to differentiate in vitro or the cells may be incorporated into a developing blastocyst in which the ES cells will contribute to all differentiated tissues of the resulting animal. Vectors for transforming ES cells and suitable genes for use as reporters and selectors are also well known.
Gene entrapment strategies have been employed to identify developmentally regulated genes. One type of entrapment vector is called a xe2x80x9cpromoter trapxe2x80x9d, which consists of a reporter gene sequence lacking a promoter. Its integration is detected when the reporter is integrated xe2x80x9cin-framexe2x80x9d into an exon. xe2x80x9cGene trap vectorsxe2x80x9d target the much more prevalent introns of the eucaryotic genome. The latter vectors consist of a splice-acceptor site upstream from a reporter gene. Integration of the reporter into an intron results in a fusion transcript containing RNA from the endogenous gene and from the reporter gene sequence.
Gene trap vectors may be made more efficient by incorporation of an internal ribosomal entry site (IRES) such as that derived from the 5xe2x80x2 non-translated region of encephalomyocarditis virus (EMCV). Placement of a IRES site between the splice acceptor and the reporter gene of a gene trap vector means that reporter gene product need not be translated as a fusion product with the endogenous gene product, thereby increasing the likelihood that integration of the vector will result in expression of the reporter gene product.
Examples from the literature of the use of promoter and gene trap vectors as well as such vectors including an IRES sequence, are listed below. Some examples involve the identification of developmentally regulated or tissue specific events making use of ES cell lines.
1. Canadian Patent application no. 2,166,850 (open for public inspection Jul. 11, 1996) Vectors and the Use Thereof for Capturing Target Genes: describes the use of transmembrane sequence encoding gene trap vectors to isolate and identify secretory proteins. Also see U.S. Pat. No. 5,767,336 issued Jun. 16, 1998.
2. U.S. Pat. No. 5,364,783 issued Nov. 15, 1994. Retrovirus Promoter Trap Vectors: describes retroviral vectors that are used to isolate transcriptionally active chromosomal regions and to identify promoter sequences. The reporter gene is placed in the U3 or U5 control region of the retrovirus.
2. Gossler, A., et al. (1989). Mouse Embryonic Stem Cells and Reporter Constructs to Detect Developmentally Regulated Genes. Science 244:463-465: describes the use of enhancer trap gene trap vectors for use in identifying developmentally regulated genes. The gene trap vector consists of the mouse En-2 splice acceptor upstream from lacZ (reporter) and a selector gene (hBa-neo).
3. Von Melchner, H., et al. Isolation of Cellular Promoters by Using a Retrovirus Promoter Trap. Proc. Natl. Acad. Sci. USA 1990, 87:3733-3737.
3. MacLeod, D., et al.: A Promoter Trap in Embryonic Stem (ES Cells Selects for Integration of DNA into CpG Islands. Nucleic Acids Res. 1991, 19:17-23.
4. Reddy, S., et al.: Retrovirus Promoter-Trap Vector to Induce lacA Gene Fusions in Mammalian Cells. J. Virol. 1991, 65:1507-1515.
5. Brenner, D. G., et al.: Analysis of Mammalian Cell Genetic Regulation in Situ by Using Retrovirus-Derived Portable Exons Carrying the Escherichia coli lacZ Gene. Proc. Natl. Acad. Sci. USA. 1989, 86:5517-5521.
6. Kerr, W. G., et al.: Transcriptional Defective Retroviruses Containing lacZ for the in Situ Detection of Endogenous Genes and Developmentally Regulated Chromatin. Cold. Spring. Harb. Symp. Quant. Biol. 1989, 54:767-776.
7. Friedrich, G. and Soriano, P.: Promoter Traps in Embryonic Stem Cells: A Genetic Screen to Identify and Mutate Developmental Genes in Mice. Genes. Dev. 1991, 5:1513-1523.
8. Skarnes, W. C., et al.: A Gene Trap Approach in Mouse Embryonic Stem Cells: The lacZ Reporter is Activated by Splicing, Reflects Endogenous Gene Expression, and is Mutagenic in Mice. Genes Dev, 1992, 6:903-918: describes gene trapping that results in activation of lacZ by splicing to endogenous exons and production of a fusion protein whose expression pattern mimics that of the endogenous gene. The resulting integration and fusion is mutagenic. Using 5xe2x80x2 RACE, the endogenous gene activated with three lacZ-based gene-trap insertions was cloned and the effectiveness of the En-2 splice acceptor site was demonstrated. For two insertions, the pattern of lacZ expression in embryos was shown to match the normal distribution of endogenous transcripts. Two of the three insertions tested cause phenotypic abnormalities in mice. One of those was an insertion into a novel gene expressed widely during development that causes perinatal death in homozygous animals. The other is an insertion into a zinc-finger gene expressed in neural cells that results in mild growth retardation after birth.
9. Von Melchner, H., et al.: Selective Disruption of Genes Expressed in Totipotent Embryonal Stem Cells. Genes. Dev. 1992, 6:919-927: where sequences upstream of nine retroviral promoter-trap insertions were cloned using if inverse PCR. Flanking probes from five ES cell lines detected transcripts, and one clone is identified as the REX-I transcription factor. Two of four lines transmitted to the germline caused embryonic-lethal phenotypes.
10. Sheriden, U., et al.: Transcriptionally Active Genomic Regions are Preferred Targets for Retroviral Integration. Mol. Cell. Biol. 1990, 64:907-912.
11. Vijaya, S., et al.: Acceptor Sites for Retroviral Integrations Map Near DNase 1-Hypersensitive Sites in chromatin. J. Virol. 1986, 60:683-692.
12. Rohdewold, H., et al.: Retrovirus Integration and Chromatin Structure: Moloney Murine Leukemia Proviral Integration Sites Map near DNAse I Hypersensitive Sites. J. Virol 1987, 61:336-343.
13. Boggs, S. S., et al.: Efficient Transformation and Frequent Single Site, Single Copy Transcription of DNA can be Obtained in Mouse Erytholeukemia Cells Transformed by Electroporation. Exp Rematol 1986, 149:988-994.
14. Soininen, R., et al.: The Mouse Enhancer Trap Locus I (Etl-1): A Novel Mammalian Gene Related to Drosophila and Yeast Transcriptional Regulator Genes. Mech Dev 1993, 39:111-123.
15. Niwa, H., et al.: An Efficient Gene-Trap Method using Poly A Trap Vectors and Characterization of Gene-Trap Events. J. Biochem 1993, 113:343-349.
16. Breindl, J. K., et al.: Retrovirus-Induced Lethal Mutation in Collagen I Gene of Mice is Associated with Altered Chromatin Structure. Cell 1984, 38:9-16.
17. Jahner, D. and Jaenisch, R.: Retrovirus-Induced de Novo Methylation of Flanking Host Sequences Correlates with Gene Inactivity. Nature 1985, 315:594-597.
18. Kratochwil, K.: Retroviral-Induced Mutation in Mov 13 Mice Affects Collages I Expression in a Tissue-Specific Manner. Cell 1989, 57:807,816.
19. Goebl, M. G. and Petes, T. D.: Most of the Yeast Sequences are Not Essential for Cell Growth and Division. Cell 1986, 46:983-922.
20. Wilson, C., et al.: P-element Mediated Enhancer Detection: An Efficient Method for Isolating and Characterizing Developmentally Regulated Genes in Drosophila. Genes. Dev. 1989, 3:1301-1313.
21. Wood, S. A., et al.: Non-Injection methods for the Production of Embryonic Stem Cell-Embryo Chimeras. Nature 1993, 365:87-89: describes time-saving methods for generating ES cell derived chimeras by morulae aggregation. These can be used to more easily carry out a prescreen based on embryonic expression of the reporter.
22. Doetschman,. T. C.: The in Vitro Development of Blastocyst-Derived Embryonic Stem Cell lines: Formation of Visceral Yolk Sac, Blood Islands and Myocardium. J. Embryo. Exp. Morph. 1985, 97:27-45.
23. Risau, W., et al.: Vasculoaenesis and Angiogenesis in Embryonic-Stem-Cell Derived Embryoid Bodies. Development 1988, 102:471-478.
24. Wiles, M. V. and Keller, G.: Multiple Hematopoetic Lineages Develop from Embryonic Stem (ES) Cells in Culture. Development 1991, 111:259-267.
25. Rossant, J., et al.: A large Scale Gene Trap Screen for Insertional Mutations in Developmentally Regulated Genes in Mice. Genetics 1995, 139:889-899: describes the use of a gene trap vector to mutagenize ES cells and the screening of ES clones by injection into blastocysts and assaying for reporter gene expression in 8,5 dpc chimeric mouse embryos.
26. Thompson, et al.: Gene Trapping in Differentiating Cell Lines: Regulation of the Lysosomal Protease Cathepsin B in Skeletal Myoblast Growth and Fusion: describes the use of a retroviral promoter trap vector in the myoblast cell line C2C12, to identify and isolate skeletal muscle specific genes.
27. Shiria, et al.: A Gene Trap Strategy to Identify Genes That Are Expressed in the Developing Mouse Nervous System. Zoological Science 1996, 13:277-283: describes use of a gene trap vector called pSneoNlacZA in ES cells. ES clones were allowed to differentiate in vitro in a suspension culture system into neurons, neuro-glia precursors cells. Neuronal cells were identified by using antibodies reactive against neuron specific proteins. Three clones that were immunohistochemically positive and xcex2-gal positive were identified.
28. Imai, et al.: Cloninc of Retinoic Acid Induced Gene, GT1, in Embryonal Carcinoma Cell Line P19: Neuron Specific Expression in Mouse Brain. Molecular Brain Research. 1995, 31:1-9: describes the use of a gene trap vector in mouse P19 embryonal carcinoma cell line which can be reproducibly differentiated to neurons and glial cells upon treatment with retinoic acid. Assay for up or down regulation of xcex2-gal activity and immunohistochemistry showed localization in neurons.
29. Forrester, et al.: An Induction Gene Trap Screen in Embryonic Stem Cells: Identification of Genes that Respond to Retinoic Acid in vitro. Proc. Nat. Acad. Sci. USA 1996, 93:1677-82: where a gene trap vector was used in ES cells and treated with retinoic acid, with a screen for genes that are induced or repressed upon retinoic acid exposure.
30. Hill, D. P. and Wurst, W. (1993): Screening for Novel Pattern Formation Genes Using Gene Trap Approaches, Methods in Enzymology 225:664-681.
31. Kim, D. G., et al. (1992): Construction of a Bifunctional mRNA in the Mouse by Using the Internal Ribosomal Entry Site of the Encephalomyocarditis Virus. Molecular and Cellular Biology, 12: 3636-3634: describes construction of vectors comprising a promoter driven selector (eg. neo) followed by EMCV 5xe2x80x2 non-translated region and either lacZ or cat, and transfection of murine ES cells with the vectors. The IRES of the EMCV permitted more efficient translation of the reporter in resulting chimeric embryos which also expressed the selection gene.
32. Chowdhury, K. et al. (1997): Evidence for the Stochastic Integration of Gene Trap Vectors into the Mouse Germline. Nucleic Acids Research, 25: 1531-1536: describes construction of a gene trap vector comprising an EN-2 splice acceptor followed by an IRES sequence and B geo (lacZ reporter fused with neo selector). Murine ES cells were transfected and neomycin resistant colonies were monitored for xcex2-galactosidase activity indicative of lacZ expression Molecular cloning of trapped exons was carried out using 5xe2x80x2-RACE.
33. D. P. Hill and W. Wurst (1993): Screening for Novel Pattern Formation Genes Using Gene Trap Approaches; Methods in Enzymology 225: 664-681.
34. G. Friedrich and P. Soriano (1993): Insertional Mutagenisis by Retroviruses and Promoter Traps in Embryonic Stem Cells; Methods in Enzymology 225: 681-701.
35. W. C. Skarnes (1993): The Identification of New Genes: Gene Trapping in Transgenic Mice; Current Opinion in Biotechnology 4: 684-689.
Regardless of the model, the process of identifying and manipulating tissue specific genes requires elaborate screening procedures to link a mutation to a particular spacial/temporal scheme or event whereby the mutation is detected in the relevant tissue. It is therefore desirable to make screening and manipulation of new spacial/temporally regulated genes in all eucaryotic models more efficient.
This invention makes use of known genes whose expression is restricted to specific tissue, tissues or specialized cells (xe2x80x9crestricted expressionxe2x80x9d) to facilitate identification and manipulation of new genes and their associated transcription control elements which have similar patterns of expression.
Accordingly, this invention provides a method of detecting a target gene having restricted expression in a eucaryotic organism, which comprises the steps of:
(i) transforming a eucaryotic cell with a DNA sequence encoding a first indicator component under the control of a promoter having restricted expression;
(ii) transforming the cell of (i) or a descendent of the cell of step (i), by operably integrating into the cell""s genome DNA lacking a promoter but which comprises a sequence encoding a second indicator component;
(iii) producing tissue or specialized cells from the cell of (ii); and
(iv) monitoring the tissue or specialized cells of (iii) for a detectable indicator resulting from both the first and second indicator components.
This invention also provides a method of obtaining a gene, a part of a gene, transcription control element or other nucleotide sequence, having restricted expression which includes isolating endogenous DNA flanking of the sequence encoding the second indicator component from a cell or descendent of a cell prepared as described above in which the detectable indicator is found. This invention also provides a method of modifying the gene, transcriptional control element or other nucleotide sequence obtained as described above which includes modification of the endogenous DNA which flanks the coding sequence of the second indicator component.
This invention also provides a method of providing a eucaryotic organism producing a detectable indicator in a specialized cell or tissue of the organism which includes growing a multi-cellular eucaryotic organism from a cell or cell descendent from a cell prepared as described above, in which the detectable indicator is found. The organism may also express in the specialized cell or tissue, the product of heterologous DNA expressed with DNA flanking of the second indicator component of the detectable indicator.
This invention makes use of a gene entrapment strategy whereby a detectable indictor is detected when more than one component of the indicator is expressed in the same cell. One method of this invention involves stable and operable insertion of a nucleic acid construct which includes one indicator component of the complementation trap under the control of a known promoter having restricted expression into the genome of the cell. This is followed by further transformation of the cell, or a cell derived from the previously transformed cell, by insertion into the cell""s genome of a trap vector which includes a DNA which encodes a second indicator component. Insertion of the trap vector into an endogenous gene may result in the transcription of both the endogenous gene and the second indicator component under control of the endogenous promoter and subsequent translation of the second component. If the trap vector integrates into an endogenous gene which is expressed in the same cell or tissue type as the first indicator component, a indicator resulting from the combination of the first and second indicator components may be detected. Thus, this invention allows one to distinguish target eukaryotic genes which are expressed in the same cell or tissue type as the promoter controlling the first component of the entrapment system. This system, as embodied by this invention is termed a xe2x80x9ccomplementation trapxe2x80x9d.
This invention provides a DNA construct comprising a splice acceptor upstream of a sequence encoding a component of a detectable indicator according to this invention. The component may be, for example, an enzyme fragment or subunit, or an enzyme functional in a pathway leading to production of a detectable indicator. This invention also provides a DNA construct comprising a promoter having restricted expression upstream of a sequence encoding a component of a detectable indicator as described above.
This invention also provides a first DNA construct comprising a splice acceptor upstream of a sequence encoding a first peptide selected from the group consisting of an alpha peptide and an omega peptide of xcex2-galactosidase (xcex2-gal), wherein the first peptide lacks xcex2-gal activity but is capable of alpha complementation to produce active xcex2-gal. This invention also provides a eucaryotic cell and a non-human, multi-cellular eucaryotic organism having operably and stably incorporated into its genome, the first DNA construct described above. This invention also provides a kit including the first DNA construct and, a second DNA construct comprising a promoter having restricted expression upstream of a sequence encoding a second xcex2-gal peptide selected such that the second peptide is capable of alpha complementation with the first peptide. This invention also provides a eucaryotic cell and a non-human, multi-cellular eucaryotic organism having operably and stably incorporated into its genome the first DNA construct and which also expresses the second DNA construct.
Trap vectors useful for this invention, including the above-described first DNA construct, may also contain additional elements such as an internal ribosome entry sequence (IRES) upstream of the second indicator component (eg. the first peptide) or, sequences that allow for directional integration of a heterologous gene, such as a recognition site for site specific recombination (eg. lox)
This invention may be used to identify tissue or cell type specific genes. For example, a nucleic acid construct containing the coding sequence of a first indicator component under control of a known tissue specific promoter is xe2x80x9cseededxe2x80x9d into cells which may be made to differentiate or whose nuclear material may be incorporated into cells which are capable of differentiation. The second component is then introduced into a cell having the xe2x80x9cseededxe2x80x9d construct, by means of the trap vector. The resulting cells, or cells derived from those cells (eg. progeny), are then induced to differentiate (for example by addition or withdrawal of a chemical inducer/repressor) or allowed to spontaneously differentiate. The cells are then screened for activity of the indicator which will occur in the cell or tissue type in which the promoter is functional.
This invention is particularly useful for screening cell or tissue specific genes or transcriptional control elements such as promoters and enhancers, in any animal from which embryonic stem (ES) cell lines may be obtained. The ES cell is subjected to the complementation trap process described above. The ES cells are then allowed to differentiate in vitro and cells are screened for the indicator. ES cells demonstrating expression of the indicator may be introduced into a blastocyst to produce an animal which will exhibit the same pattern of expression. Where introduction of the trap vector results in disruption of the target gene, the animal resulting from the mutated ES cells will be useful as a xe2x80x9cknock-outxe2x80x9d animal useful for study of the target gene. Alternatively, location of the trap vector into the endogenous gene will provide means for subsequent disruption of the target gene for production of xe2x80x9cknock-outxe2x80x9d animals. The trap vector of this invention may be used as a means for locating, cloning, sequencing, and further mutation of the target gene or promoter or enhancer sequences associated with the target gene.
This invention will make new transcriptional control elements available for use in making new transgenic animals displaying cell, tissue or organelle specific expression of transgenes. Promoters made available by use of this invention may themselves be made to control the first indicator component in the complementation trap of this invention in a xe2x80x9cleap-frogxe2x80x9d procedure whereby new genes having the same restriction pattern as the promoter or may be located.